Background

Caloric restriction (CR) consists in the limitation of food intake below the ad libitum level without malnutrition and is one of the most consistent non-pharmacological interventions for increasing life expectancy and protecting against the deterioration of biological functions [1]. Studies in several animal models have shown that CR decreases or prevents the progression of age-related conditions, such as cardiovascular and neurodegenerative diseases [2, 3].

With advancing age, there is an increased oxidative stress, defined as an imbalance between cellular production of reactive species and antioxidant defenses [3]. Excess of reactive species can cause damage to lipids, proteins, and DNA and can be the cause of numerous neurodegenerative diseases [4]. The hippocampus plays an essential role in learning and memory and is extremely vulnerable to oxidative stress during the developmental stage, due to a diminished ability of neurons to maintain the redox homeostasis [5]. Therefore, the maintenance of a balanced redox state in the hippocampus is important to prevent cognitive decline and can be more vulnerable during the developmental stage. This led us to hypothesize that CR would be more beneficial in rats the earlier it was started.

In order to protect itself from the damage caused by reactive species, the body has a number of defense mechanisms, including the reduced glutathione (GSH), the most important non-enzymatic antioxidant, as well as the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). These antioxidant defenses are regulated in response to the level of reactive oxygen species (ROS) production and lipid peroxidation [6]. It is noteworthy that the brain is especially susceptible to oxidative stress due to its high oxygen turnover and to its high content of polyunsaturated fatty acids [7]. Another important enzyme involved in the protection against lipid peroxidation is paraoxonase 1 (PON1). It has been demonstrated that PON1 may act as a neuroprotective agent against oxidative stress, since lower PON1 activity has been associated to increased incidence of Alzheimer’s and Parkinson’s diseases, as well as dementia [8–10].

The benefits of CR on the oxidative stress in the brain, and especially on the protection against age-related neurodegenerative diseases, have been the subject of several studies [11, 12]. Therefore, understanding the biochemical changes during CR is essential for the development of additional therapeutic interventions for the prevention of such diseases. Considering that the effects of CR in the central nervous system are still poorly understood and most studies were carried out in mature animals, the present study aimed to investigate whether the age at onset of CR could differently affect the redox status in the rat hippocampus.

Methods

Animals and experimental design

Thirty-two male Wistar rats, 35 days old (35d; n = 16) and 65 days old (65d; n = 16) were fed ad libitum or subjected to 30 % CR for 12 weeks. The animals were maintained under temperature, humidity and light-controlled conditions (22 ± 2 °C, 65–75 %, 12 h light/12 h dark cycle). The experiments were conducted according to the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and approved by the Ethics Committee on Animal Experimentation from the Federal University of Pelotas (process no. 23110.009827/2012-89/CEEA 9827). The rats were weighed and randomly divided into four groups (n = 8/age/group: 35d control, 65d control, 35d CR, 65d CR). All rats received standard laboratory chow (PuroTrato® - Pure Lab 22, Brazil; 31.7 % carbohydrate, 22.1 % protein, and 15.9 % fat). During the first 5 days, rats were adapted to the diet and intake was measured. After that, the CR was progressive, with 10 % restriction in the first week, 20 % in the second, and 30 % from the third week until the end of the experiment. Food intake was monitored daily, and rats were weekly weighed. The measurement of the nasoanal length [NAL (cm)] was performed at the end of the experiment, and the Lee index was calculated from the ratio between the cubic root of body weight and the nasoanal length of the animal [∛Weight (g)/NAL (cm)].

Object recognition test

The object recognition test was performed according to the methodology previously described [13]. Twelve weeks after the beginning of the controlled feeding, rats were placed in an open-field arena consisting of a wooden box (size 40 × 50 cm and 50 cm high) surrounded by a glass wall. All rats were submitted to an adaptation session and allowed to freely explore the object free open field for 5 min. Twenty-four hours after the open-field exploration, rats were trained and tested in a novel object recognition task. Training in the object recognition task took place in the same arena used for the open field. The object recognition test required that the rats recalled which of two plastic objects they had been previously familiarized with. Twenty-four hours after arena exploration, training was conducted by placing individual rats into the field, in which two identical objects (A1 and A2) were positioned in two adjacent corners, 10 cm from the walls. The test session occurred 24 h after the training and the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B) object. All objects presented similar textures, colors, and sizes, but distinctive shapes. A recognition index calculated for each animal was calculated by the ratio TB/(TA + TB) (TA = time spent exploring the familiar object A; TB = time spent exploring the novel object B). Between each trial, the objects were washed with 10 % ethanol solution. Exploration was defined as sniffing or touching the object with the nose and/or forepaws.

Blood collection and analysis

At the end of the experiment (after 12 weeks of treatment), rats were fasted overnight and euthanized by decapitation. Blood samples were collected without anticoagulant, incubated at room temperature (25 °C), and centrifuged at 1000×g for 10 min. Serum was harvested and stored at −20 °C until analysis. Biochemical analyses were performed using a spectrophotometer (Cirrus 80 MB®, Femto). Serum samples were analyzed according to manufacturer recommendations for total protein (biuret method, Bioclin®), albumin (bromocresol method, Bioclin®), urea (UV Urease, Bioclin®), creatinine (alkaline picrate, Bioclin®), glucose (glucose oxidase, Doles®), total cholesterol (cholesterol esterase, cholesterol oxidase and peroxidase, Life Biotecnologia®), triglycerides (Trinder, Life Biotecnologia®), HDL cholesterol (indirect Doles®). The LDL cholesterol concentration was estimated according to previously described [14].

Hippocampus dissection

After euthanasia, the brain was removed and the hippocampus was dissected and placed in saline/phosphate-buffered solution (PBS pH 7.4). The hippocampus was weighed, placed in a microtube, and stored in liquid nitrogen until analysis. Immediately after thawing, the hippocampus was cut in cross-sections of 0.3 mm using a McIlwain Tissue Chopper and placed in PBS (pH 7.4).

GSH concentrations

GSH content was determined according to the method previously described [15]. The hippocampal slices were homogenized in sodium phosphate buffer (0.1 M, pH 8.0) containing 5 mM EDTA, and the proteins were precipitated with 1.7 % metaphosphoric acid. The supernatant was then incubated with o-phthaldialdehyde (1 mg/mL of methanol) at room temperature for 15 min. Fluorescence was measured using excitation and emission wavelengths of 350 and 420 nm, respectively. A calibration curve was performed with standard glutathione solutions (0–500 mM). The GSH concentrations were expressed as nmol/mg protein.

GPx activity

GPx activity was measured in the hippocampus as previously described [16] using tert-butyl hydroperoxide as substrate. GPx activity was determined by monitoring NADPH (0.1 mM) disappearance at 340 nm in a medium containing 2 mM GSH, 0.15 U/mL glutathione reductase, 0.4 mM azide, and 0.5 mM tert-butyl-hydroperoxide. One GPx unit was defined as 1 μmol of NADPH consumed per minute, and the specific activity is represented as U/mg protein.

SOD activity

SOD activity in the hippocampus was assessed by monitoring the inhibition of superoxide-dependent adrenaline auto-oxidation in a spectrophotometer at 480 nm using the method previously described [17]. Results were expressed as U/mg protein.

Evaluation of intracellular ROS production

Intracellular ROS production was measured using the non-fluorescent cell permeating compound 2′-7′-dichlorofluorescein diacetate (DCF-DA). Hippocampus samples were homogenized in PBS (pH 7.4) with 140 mM KCl and treated with DCF-DA (10 mM) for 30 min at 37 °C. The fluorescence was then measured in a plate reader (Spectra Max GEMINI XPS, Molecular Devices, USA) at 485 nm excitation and at 520 nm emission, as previously described and adapted for evaluation of tissue slices [18]. Values were expressed as unit of fluorescence/mg protein.

TBARS measurement

Formation of malondialdehyde (MDA), resulting from lipid peroxidation, was measured by the quantification of thiobarbituric acid-reactive substances (TBARS) which was adapted from previously described methods [19]. Briefly, 100 μL of tissue homogenate was added to 200 μL of trichloroacetic acid 10 % and 300 μL of 0.67 % TBA in 7.1 % sodium sulfate and placed in a boiling water bath for 1 h. Immediately after, the mixture was placed in cold water for 5 min. The resulting pink-stained complex was extracted with 400 μL of butyl alcohol, and the samples were centrifuged at 5000×g for 5 min. The organic phase fluorescence was measured using excitation and emission wavelengths of 515 and 553 nm, respectively. A calibration curve was performed with 1,1,3,3-tetramethoxypropane. Results were expressed as nmol of TBARS/mg protein.

NO production

Nitric oxide (NO) metabolites, nitrate (NO3 −) and nitrite (NO2 −), were determined according to a methodology previously described [20]. Briefly, homogenates from hippocampal slices were mixed with 25 % TCA and centrifuged at 1800×g for 10 min. The supernatant was immediately neutralized with 2 M potassium bicarbonate. Nitrate was reduced to nitrite by the addition of nitrate reductase. Afterwards, the total nitrite was measured by a colorimetric assay at 540 nm, based on the Griess reaction. A standard curve was performed using sodium nitrate (0–80 mM). Results were expressed as mM of nitrite/mg protein.

PON1 activity

The activity of PON1 was measured by its arylesterase activity as previously established [21]. The arylesterase activity was measured by the phenol formation rate through monitoring the increase in absorbance at 270 nm at 25 °C. The working reagent consisted of 20 mM Tris/HCl, pH 8.0, containing 1 mM of CaCl2 and 4 mM phenylacetate as substrate. The samples were diluted 1:3 in a 20-mM Tris/HCl buffer and were added to the working reagent and the change in absorbance recorded for 60 s. The activity was expressed in kU/L, based on the phenol extinction coefficient.

Tissue protein content

Total tissue protein content was determined by the modified method of Lowry as previously described [22], using bovine serum albumin as a standard.

Statistical analysis

Data is reported as mean ± standard error mean (SEM). All analyses were performed using GraphPad Prism 5 (GraphPad®, La Jolla, CA, USA). Two-way ANOVA was used to test the effects of the diet, age at the beginning of the experiment, and the interaction between age and diet. When the interaction was significant, t test was performed between individual groups. p values below 0.05 were considered significant.

Results

Effect of CR diet on body weight and serum biochemistry

Body weight declined by 16 and 21 % for 35d and 65d rats on CR diet, respectively, when compared to its controls (p < 0.05; Fig. 1). There were no significant differences between CR groups and controls for Lee index (diet p = 0.961; data not shown). Urea, total cholesterol, triacylglycerol, HDL cholesterol, and LDL cholesterol were lower in CR 35d rats than in control 35d rats (p < 0.05; Table 1). On the other hand, only urea and triacylglycerol were lower in CR 65d rats in comparison with control 65d rats (Table 1), whereas LDL cholesterol was higher in CR- than in control 65d rats (p < 0.05; Table 1). Other serum parameters were not different between groups (p > 0.05; Table 1).

Fig. 1
figure 1

Body weight gain of control (solid lines) and calorie restriction (CR)-fed rats (dashed lines) throughout 12 weeks with the onset of CR at 35d or 65d ages

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Table 1 Serum biochemistry of 35d and 65d rats fed ad libitum (control) and caloric-restricted diet (CR) for 12 weeks
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Object recognition test

No differences were detected between CR groups and controls in the object recognition task (p > 0.05; Fig. 2), indicating that neither CR nor age affected the memory of the rats.

Fig. 2
figure 2

Object recognition index after 12 weeks with the onset of CR at 35d or 65d ages

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Effect of CR diet on oxidative stress parameters in the hippocampus

There were no significant differences among groups for reactive oxygen and nitrogen species production (Fig. 3a, e, respectively), neither in lipid peroxidation (Fig. 3f) which is a common consequence of oxidative stress. However, GSH, an important antioxidant defense mainly in brain tissues, was higher in CR-fed rats (p = 0.001; Fig. 3b) and in 65d rats compared to 35d rats (p = 0.036; Fig. 3b). GPX activity, an enzyme that reduces lipid hydroperoxides and is GSH dependent, was higher in 65d rats only regardless of the diet (Fig. 3d). SOD activity, an enzyme that catalyzes superoxide radicals dismutation, was not influenced by age or diet in this model (Fig. 3c).

Fig. 3
figure 3

Oxidative stress parameters, DCF (a) , GSH (b), SOD (c), GPx (d), NO (e) and TBARS (f), in the rat hippocampus throughout 12 weeks with the onset of CR at 35d or 65d rats

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Discussion

The brain plays a key role in managing energy homeostasis and is an important target of dietary interventions. In this study, we verified that 65d and 35d rats, when kept under moderate CR, may exhibit different biological responses. Our results shown that when CR is started in 35d or 65d rats, it can still sustain weight gain and growth, although slower than in the respective control groups, and had no detrimental effects to the nutritional status. On the other hand, CR reduced serum lipids more in 35d than in 65d rats, since the latter had an unfavorable lipid profile with increased LDL and decreased HDL levels. Possibly, CR started in 35d rats can promote a better adaptation to the imposed metabolic challenge than when started in 65d rats. However, it is feasible that the positive effects of CR in 65d rats previously described [1, 23] may outweigh the increased LDL observed in the current study. Consequently, we could expect a reduction of the oxidative damage to lipids and a reduced atherogenic potential of LDL in CR rats.

In this study, CR did not affect serum PON1 activity when started in 35d or 65d rats. This indicates that although CR affected serum HDL and LDL cholesterol concentration, it was not detrimental for PON1 activity. As stated before, PON1 has a role in protecting cell membrane against lipoperoxidation [24]. Therefore, the maintenance of high levels of PON1 activity can be beneficial to protect neuronal cells against oxidative stress. Incidence of amyotrophic lateral sclerosis, a disease linked to motor neuron damage mainly due to a mutation in the SOD1 gene, was lower in individuals with genetic mutations associated to higher levels of serum PON1 activity [24]. This evidence points to a role of oxidative stress in neuronal damage and PON1 as a protective factor. Therefore, the absence of changes in PON1 activity in CR-fed mice can be beneficial, indicating no reduction in cell membrane protection.

There were no differences in the level of ROS, NO content and lipid peroxidation among groups, despite previous reports indicating the opposite [23, 25]. This can be due to the short-term CR (12 weeks) used in this study. Despite that, GSH content, which plays a crucial role protecting the brain against the oxidative stress, was higher in CR rats. In addition, GPx activity and GSH content were also higher when CR was started in 65d rats. A study evaluating the antioxidant system in mitochondria from young and old rats indicated that GPx activity and GSH content, but not SOD, increased with age [25], in agreement with our current findings. When the antioxidant parameters were measured in our study, the 35d rats were 119 days old and the 65d rats were 149 days old, indicating that this period of development may be critical for the enhancement of hippocampus GPx activity, since its activity was twice higher in older rats. Furthermore, CR increased GSH content when started in both 35d and 65d rats and was overall higher in 65d rats. Corroborating with our results, a previous study also showed an increased GSH content in the hippocampus of rats subjected to CR [26]. The improvement in 65d rats can be related to the higher body weight loss observed in this group. Therefore, our findings demonstrated that CR can improve the antioxidant status of the rat hippocampus, and although the redox profile was changed with age alone, only GSH content was increased by age and diet simultaneously.

Recently, it was suggested that a 12-week CR diet in humans can improve memory and prevent age-related decline and its positive effects are more related to the weight loss rather than to an overall reduced body weight [27]. Our results suggested no loss or improvement in memory of rats subjected to CR independent of age. Different from the data reported for humans, in our study, mice were still in the growth phase and CR did not lead to weight loss, but rather to a reduction in body weight gain. This may explain why the 12-week CR in our study did not affected memory, although it affected the redox status of the hippocampus.

Conclusions

In conclusion, rats subjected to 12 weeks of caloric restriction had improved hippocampus GSH content, which was more pronounced when CR was started at 65d rats. GPx activity was only increased with age. Despite that, CR did not affect other hippocampus antioxidant enzymes and ROS level. Calorie restriction did not have any positive or negative effect on memory and nutritional status when started in both 35d and 65d rats.